Note: Descriptions are shown in the official language in which they were submitted.
WO 2021/188379
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PRODUCTION OF SOLUBLE RECOMBINANT PROTEIN
Rights in the Invention
The invention was made with United States Government support under Grant No.
1R43A1148018-01A1 FAIN: R43AI148018, awarded by the National Institutes of
Health, and the
U.S. Government has certain rights in the invention.
Reference to Related Applications
This application claims priority to U.S. Provisional Application No. 63/152,
954 filed
February 24, 2021; U.S. Provisional Application No. 62/990,083 filed March 16,
2020; and U.S.
Application No. 16/819,775 filed March 16, 2020, and presently pending, the
entirety of each of
which is specifically incorporated by reference.
Field of the Invention
The invention is directed to methods and compositions to express and purify
products such
as peptides and proteins in microorganisms.
In particular, pre-products are expressed
recombinantly, wherein the cytoplasm of the microorganism alters the expressed
pre-products to
produce products in a final or usable form. Alterations include shifting of
the redox state of the
cytoplasm and site-directed cleavage and/or ligation.
Description of the Background
E. coli is a widely used host to produce recombinant proteins for research and
therapeutic
purposes. Recombinant proteins can be expressed in E. coli cytoplasm or
periplasm. One limitation
to cytoplasmic recombinant protein expression in E. coil is that, to initiate
expression of
recombinant protein in E. coli, the coding sequence of the protein should
start with the ATG codon,
which is translated to formyl-methionine and then processed by
formylmethioninc deformylase to
become N-terminal methionine. Therefore, for recombinant protein expression in
E. coli, the ATG
codon is added to the native or mature protein sequence. During intracellular
expression of
recombinant protein, the N-terminal Methionine is usually excised by
endogenous E. coli
methionine aminopeptidase (MAP). This process is not necessarily efficient for
recombinant
proteins, even if the residue adjacent is optimal for cleavage, likely due to
overexpression of the
recombinant protein and the limited amount of MAP present. As a result, a
substantial amount of
the recombinant protein may have Methionine as the first amino acid. This is
undesirable for most
proteins as the N-terminal Methionine is not a part of the mature protein
sequence. The presence
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of the N-terminal Methionine may also cause structural changes to a protein
that affects its
function. Existing methods to ensure effective cleavage of the N terminal-
methionine include in
vitro treatment with recombinant MAP. Another approach is adding the MAP
coding sequence to
the expression vector and thus co-expressing MAP along with the recombinant
protein. In the latter
case, co-expression of MAP may reduce expression of the desired recombinant
protein. Both
approaches are time-consuming and costly to implement. Thus, it would be
desirable to have an
E. coli expression strain where MAP was highly expressed without significantly
inhibiting the
recombinant protein expression. This would allow the production of increased
amounts of the
target protein with its native sequence in the cytoplasm.
Recombinant proteins expressed in the cytoplasm of E. coli may form insoluble
inclusion
bodies. Proteins in inclusion bodies may be refolded in-vitro to form soluble
proteins. These
proteins will contain an N-terminal methionine, which is undesirable.
The E. coli cytoplasm has a reducing environment, and recombinant proteins
containing
disulfide bonds are usually insoluble when expressed intracellularly. In
contrast, the periplasm of
E. coli has an oxidative environment. Therefore, many recombinant proteins
containing disulfide
bonds are secreted into the periplasm in order to ensure proper folding and
solubility. The signal
peptide that directs recombinant protein into periplasm is clipped off during
the secretion process,
resulting in the production of protein with the native amino acid sequence.
However, the
translocation mechanisms that direct proteins to the periplasm have limited
capacity, and so
periplasmic expression level of recombinant proteins is usually low. On the
other hand, expression
in the E. coli cytoplasm can lead to grams of recombinant proteins per liter
of cell culture.
Therefore, it would be desirable to be able to express soluble, properly
folded disulfide-bonded
proteins in the cytoplasm. Furthermore, it would be desirable if these
proteins could be produced
without the N-terminal Methionine.
Commercially available E. coli strains such as Origami (EMDMillipore),
Shuffle (New
England Bio) with gor-/trx- mutations, can produce soluble, intracellular
proteins containing
disulfide bonds, but these cell strains are crippled and do not grow to a high-
density, limiting
production yield. Thus, while these strains are suitable for generating
research material, their low
growth levels make them difficult to use commercially. Thus, a need exists for
strains that express
high levels of properly folded intracellular disulfide-bonded proteins that do
not contain an N-
terminal methionine.
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Summary of the Invention
The present invention overcomes the problems and disadvantages associated with
current
strategies and designs and provides new compositions and methods for producing
recombinant
peptides and proteins.
One embodiment of the invention is directed to methods of producing
recombinant
peptides and proteins in bacteria comprising: expressing the protein in a
bacteria containing an
expression vector that encodes the protein sequence including a promoter and
the bacteria also
contains a gene under the control of a promoter, which is integrated into the
genome of the host,
wherein the polypeptide expressed by that gene facilitates the expression,
folding or solubility of
the recombinant protein in the cytoplasm and isolating the protein.
Another embodiment of the invention is directed to methods of producing
recombinant
peptides and proteins in bacteria comprising: expressing the protein in a
bacteria containing an
expression vector that encodes the protein sequence including a promoter and
the bacteria also
expresses a peptidase gene, which is integrated into the genome of the host
cell, where the
peptidase expression is under the control of a promoter, such that the
peptidase acts on the protein
expressed and removes a formyl-methionine group from the N-terminal portion of
the protein; and
isolating the protein. Peptidases that remove an N-terminal methionine can be
referred to as
Methionine amino peptidases (MAP). Preferably the integrated gene contains a
ribosome binding
site, an initiation codon, and an expression enhancer and/or repressor region.
Preferably the
recombinant cell has reduced activity of only one disulfide reductase enzyme
or a reduced activity
of only two disulfide reductase enzymes. Preferably the recombinant cell is an
E. coli cell or a
derivative or strain of E. coli, and preferably the recombinant protein
expressed comprises tetanus
toxin, tetanus toxin heavy chain proteins, diphtheria toxoid, tetanus toxoid,
Pseudomonas
exoprotein A, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid,
Clostridium
perfringens toxoid, Escherichia coli heat-labile toxin B subunit, Neisseria
meningitidis outer
membrane complex, Hemophilus influenzae protein 13, Flagellin Fli C.
cytokines, single chain
antibodies, camelids, nanobodies and fragments, derivatives, and modifications
thereof. Also
preferably, the recombinant protein may be a pre-protein prorelaxin, insulin
and members of the
insulin-like family. Preferably the integrated gene and/or expression vector
contains an inducible
promoter for the peptidase. Expressing comprises inducing the inducible
promoter with a first
inducing agent and contains an expression vector that encodes the recombinant
peptide or protein
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which may be inducible with a second inducing agent. Preferably the first and
second inducing
agents are the same, although they may be different. Preferably the first
integrated gene or
expression vector contains an inducible second promoter and expressing the
peptidase comprises
inducing the inducible second promoter with the first inducing agent.
Preferably isolating
comprises chromatography wherein the chromatography comprises a sulfate resin,
a gel resin, an
active sulfated resin, a phosphate resin, a heparin resin, or a heparin-like
resin. Preferably the
isolated protein expressed is conjugated with polyethylene glycol and/or a
derivative of
polyethylene glycol or with a polymer such as, for example, a polysaccharide,
a peptide, an
antibody or portion of an antibody, a lipid, a fatty acid, a small molecule,
hapten or a combination
thereof.
Another embodiment of the invention is directed to methods of producing a
soluble or
insoluble peptide comprising: expressing the peptide with a formyl-methionine
group at an N-
terminus of the peptide from a recombinant cell containing an expression
vector that encodes the
peptide and expressing a peptidase from an integrated gene of a recombinant
cell that acts on the
peptide expressed and removes the formyl-methionine group from the N-terminus
of the peptide;
and isolating the peptide.
Another embodiment of the invention is directed to methods of producing a
peptide
comprising: expressing the peptide with a formyl-methionine group at an N-
terminus of the peptide
from a recombinant cell containing an expression vector that encodes the
peptide, wherein the
recombinant cell has a reduced activity of one or more disulfide reductase
enzymes and the
expression vector contains a promoter functionally linked to a coding region
of the peptide,
wherein the reduced activity of one or more disulfide reductase enzymes
results in a shift the redox
status of the cytoplasm to a more oxidative state as compared to a recombinant
cell that does not
have reduced activity of one or more disulfide reductase enzymes, and
expressing a peptidase from
an integrated gene of a recombinant cell that acts on the peptide expressed
and removes the formyl-
methionine group from the IN-terminus of the peptide; and isolating the
peptide. Preferably the
expression vector contains a ribosome binding site, an initiation codon, and
an expression
enhancer/repressor region. Preferably the recombinant cell has a reduced
activity of only one
disulfide reductase enzyme or only two disulfide reductase enzymes. Preferably
the one or more
disulfide reductase enzymes comprise one or more of an oxidoreductase, a
dihydrofolate reductase,
a thioredoxin reductase, or a glutathione reductase. Preferably the
recombinant cell is an E. coil
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cell or a derivative or strain of E. coli and the peptide or protein comprises
tetanus toxin, tetanus
toxin heavy chain proteins, diphtheria toxoid, tetanus toxoid, Pseudomonas
exoprotein A,
Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, Clostridium
perfringens toxoid,
Botulism toxin, Escherichia coli heat-labile toxin B subunit, Neisseria
meningitidis outer
membrane complex, Hemophilus influenzae protein D, Flagellin Fli C. cytokines,
single chain
antibodies, camelids, nanobodies, and fragments, derivatives, and
modifications thereof.
Preferably the promoter is an inducible promoter and expressing comprises
inducing the inducible
promoter with an inducing agent.
Preferably isolating comprises chromatography, wherein the chromatography
comprises a
sulfate resin, a gel resin, an active sulfated resin, a phosphate resin, a
heparin resin, or a heparin-
like resin. Preferably the peptide isolated is conjugated with polyethylene
glycol (PEG) and/or a
derivative of PEG, or coupled to a polymer such as, for example, a
polysaccharide, a peptide, an
antibody or portion of an antibody, a lipid, a fatty acid, small molecule,
hapten or a combination
thereof.
Another embodiment of the invention is directed to an E. coli cell line
containing a gor
mutation. Preferably the cell line comprises cells obtained or derived from
ATCC Deposit number
PTA-126975.
Another embodiment of the invention is directed to methods of producing a
protein
comprising: expressing a preprotein in a recombinant cell which contains a
recombinantly
engineered protease gene. Preferably the protease gene and/or the preprotein
gene contains a
promotor and/or a translation induction sequence. The promoters may be the
same of different
and the translation induction sequences, if present, may be the same or
different for the genes.
Preferably after expression of the preprotein, expression of the protease gene
is induced such that
the preprotein is cleaved to form the protein; and harvesting the protein.
Preferably the preprotein
is selected from the group consisting of pro-insulin, pro-insulin-like
proteins, prorelaxin,
proopiomelanocortin, a proenzyme, a prohormoncs, proangiotensinogcn,
protrypsinogen,
prochymotrypsinogen, propepsinogen, proproteins of the coagulation system,
prothrombin,
proplasminogen, proproteins of the compliment system, procaspases,
propacifastin, proelastase,
prolipase, procarboxypolypeptidases, proteins containing a cleavable leader
sequence, cleavable
tag sequences, proteins containing a cleavable extra N- or C-terminal amino
acid (e.g., Met).
Preferably the protease gene is integrated into the genome of the recombinant
cell. Also
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preferably, a methionine aminopeptidase gene is integrated into the genome of
the recombinant
cell, wherein expression of the methionine aminopeptidase gene removes an N-
terminal
methionine from the preprotein or the protein. The expression of the
methionine aminopeptidase
gene is preferably under the control of an inducer sequence and the inducer
sequence of the
methionine aminopeptidase and the translation induction sequence of the
preprotein may be the
same or different. Also preferably, the recombinant cell has a reduced
activity of one or more
disulfide reductase enzymes which may be and E. coli with a gor mutation.
Another embodiment of the invention is directed to a recombinant cell line
containing a
methionine aminopeptidase gene and a protease gene, both of which are
integrated. Preferably the
cell further contains a reduced activity of one or more disulfide reductase
enzymes, which may be
attributed to a gor mutation.
Other embodiments and advantages of the invention are set forth in part in the
description,
which follows, and in part, may be obvious from this description, or may be
learned from the
practice of the invention.
Description of the Invention
Proteins in E. coli are typically expressed with a methionine at the N-
terminus because the
correspondent ATG cc-)don is required for initiation of translation. Proteins
expressed
intracellularly, therefore, contain N-terminal Methionine that is not part of
the native amino acid
sequence (unless the native sequence begins with a methionine). Removal of the
N terminal
methionine by MAP can be important for the function and stability of proteins.
An endogenous
methionine aminopeptidase (MAP) can cleave the N terminal methionine of newly
synthesized
protein, typically up to 60-70%. However, in highly expressed recombinant
proteins, the level of
activities of the endogenous MAP may not be sufficient to remove a desired
amount of the N-
terminal methionine. Removing N-terminal Methionine would be a significant
issue in producing
intracellular recombinant proteins in E. coll. Thus, E. coli strains that can
efficiently cleave
unwanted N -terminal Met would be highly desirable.
Solubility and proper folding of recombinant proteins expressed
intracellularly is of a
concern for E. coli expression systems. E. coli cytoplasm has a reducing
environment that does
not favor disulfide bond forming. As a result, recombinant proteins containing
disulfide bonds
are usually insoluble when expressed intracellularly. Purification of these
insoluble proteins can
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be difficult, expensive and time consuming. High cell densities are preferable
for the production
of recombinant proteins, especially for commercial use.
Microorganisms genetically engineered to express large quantities of properly
configured
recombinant protein that also effectively remove N-terminal formyl-methionine
have been
surprisingly developed. These microorganisms are genetically engineered to
express soluble
recombinant proteins containing disulfide bonds in the cytoplasm and to remove
N-terminal
Methionine. These microorganisms produce a large quantity of the properly
folded intracellular
recombinant protein containing disulfide bonds without Methionine at the
protein's N-terminus.
Preferably, microorganisms contain one or more methionine aminopeptidase (MAP)
genes
incorporated into the genome of the bacteria. Peptidases that remove an N-
terminal methionine
include, but are not limited to, E. coli MAPs, Yeast MAPs and human MAPs, and
their mutants,
all of which can be utilized. The coding sequence of the MAP, under the
control of an inducible
promoter, was inserted into the bacterial genome, preferably in a manner as to
prevent disruption
of the genome. Having an inducible promoter allows the initiation of the
expression of additional
MAP at a selected time, preferably only when more MAP is needed to effectively
remove formyl-
methionine from overexpressed recombinant protein. The promoter for the MAP
gene can be the
same or different from the promoter used for the recombinant protein. In one
example, the tac-
promoter was utilized as a lactose/IPTG inducible promoter for both the MAP
gene and the
recombinant protein so the expression of the MAP and the recombinant protein
can be induced at
the same time. Different combinations of inducible promoters for expression of
MAP and for that
of the recombinant protein can be used to regulate the timing of the
expression of each.
Incorporation of additional MAP into the genome is particularly desirable as
the stable
bacterial expression strains created can be used for the intracellular
production of recombinant
proteins with unwanted f-Met cleaved in vivo. Previously, removal of unwanted
N terminal
methionine has been done by post translationally in vitro digestion using
purified MAP or by co-
expression of MAP in the same vector as the recombinant protein or using an
additional vector.
The process is long and complicated. By using a cell line that can cleave f-
met on demand, in
vivo, these microorganisms greatly simplify protein expression and
purification process.
MAPs cleave the N-terminal Methionine with specific requirements for the
adjacent amino
acids. The use of at least one additional MAP may allow the more efficient
production of
intracellular proteins without N terminal Methionine. If more than one MAP is
inserted, they can
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be the same or different MAP genes. The transcriptions of these MAP genes may
be under the
same or different inducible promoters. These promoters may be the same or
different from the
promoter used to express the recombinant proteins. A combination of inducible
promoters can be
used to control the timing of and the amount of the production of
intracellularly expressed
recombinant protein without N terminal methionine.
E. coli strains capable of expressing soluble, properly folded intracellular
recombinant
proteins containing disulfide bonds have been described (see U.S. Patent Nos.
10,597,664 and
10,093,704). An example of such a strain is the E. coli (BL21 Gor-). BL21Gor-
has been used to
express soluble, properly folded intracellular recombinant proteins containing
disulfide bonds at
high levels, including the vaccine carrier protein CRM197, a genetically
detoxified diphtheria toxin.
Approximately 60% of the CRM197 expressed in these cells contained an N-
terminal methionine.
The insertion of an additional MAP gene under the control of a promoter into
such a strain allows
for the production of soluble, properly folded intracellular recombinant
proteins containing
disulfide bonds and without N-terminal Methionine. An example of such a strain
is the E. coli
(BL21 Gor/Mct) strain. This strain can produce intracellularly soluble
proteins, with disulfide
bonds and without N-terminal Methionine, in grams quantity per liter of cell
culture. CR_M197
expressed in BL21 Gor/Met cells contained very low levels of N-terminal
Methionine.
Furthernaore, the incorporation of the inducible methionine aminopeptidase
gene into the E. coli
genome did not significantly affect CRM197 expression levels.
The MAP gene was inserted into the genome by homologous recombination,
although
several other options to facilitate insertion are available. The approach used
for the creation of the
Gor/Met cell strain is an example of gene insertion. For Gor/Met cells, red
recombinase system
was used to insert the MAP gene into Gor locus in BL21 Gor- cells. The MAP
gene was cut from
the BL21 genome using PCR and put under the control of Tac promoter and
downstream of a
chloramphenicol acetyltransferase (CAT) gene flanked by two short flippase
recognition target
(FRO sequences. MAP and CAT gene together formed a transfer gene cassette.
Fifty bases each
of sequences flanking upstream and downstream of the original Gor locus were
added to the
transfer cassette upstream and downstream, respectively, by PCR. The final PCR
product was
used to transform to BL21 Gor- cell line that had already transformed with Red
recombinase. The
expression of red recombinase in the cell facilitated the homologous
recombination of the
sequences flanking Gor locus with the transfer cassette. Bacterial colonies
that were resistant to
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chloramphenicol were confirmed to have successful transfer cassette insertion.
Confirmed
bacteria were subsequently transformed with flippase gene whose expression
recognized FRT
sequence flanking the CAT gene and clipped the gene away to leave the MAP
inserted. Thus, the
Gor/Met cell line has a MAP gene located at the Gor locus, without disturbing
other parts of the
genome.
To produce large quantities of protein, such as CRM197 from E. coil host
cells, an f-Met
that is present at the N-terminus of the protein is enzymatically removed in
the cytoplasm.
Production quantities are typically quantified as mg/L of bacterial cell
culture. Protein production
achieved 25 mg/L or more, 50mg/L or more 100 mg/L or more, 200mg/L or more,
300mg/L or
more, 400mg/L or more. 500mg/L or more, 600 mg/L or more, 700 mg/L or more,
800 mg/L or
more, 900 mg/L or more, 1,000 mg/L or more, 1,500mg/L or more. or 2,000mg/L or
more. Protein
expressed, as desired, include both full length and/or truncated proteins, as
well as modified amino
acid sequences of the protein. Modifications include one or more of
conservative amino acid
deletions, substitution and/or additions. A conservative modification is one
that maintains the
functional activity and/or immunogenicity of the molecule, although the
activity and/or
immunogenicity may be increased or decreased. Examples of conservative
modifications include,
but are not limited to amino acid modifications (e.g., single, double and
otherwise short amino
acid additions, deletions and/or substitutions), modifications outside of the
active or functional
sequence, residues that are accessible for conjugation in forming a vaccine,
modifications due to
serotype variations, modifications that increase immunogenicity or increase
conjugation
efficiency, modification that do not substantially alter binding to heparin,
modifications that
maintain proper folding or three dimensional structure, and/or modifications
that do not
significantly alter immunogenicity of the protein or the portions of the
protein that provide
protective immunity.
Recombinant cells used are preferably E. coil bacteria and, preferably, E.
coil that are
genetically engineered to shift the redox state of the cytoplasm to a more
oxidative state such as,
for example, by mutation of one or more disulfide reductase genes such as, for
example, an
oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase, a
glutamate cysteine lyase, a
disulfide reductase, a protein reductase, and/or a glutathione reductase.
Preferably one or more
disulfide reductase genes are mutated and rendered non-functional or
marginally functional such
that the redox state of the cytoplasm of the cell is shifted to a more
oxidative state as compared to
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wild type without compromising viability. Oxidative protein folding involves
the formation and
isomerization of disulfide bridges and plays a key role in the stability and
solubility of many
proteins including CRM197. Formation and the breakage of disulfide bridges is
generally catalyzed
by thiol-di sulfide oxidoreductases. These enzymes arc characterized by one or
more Trx folds that
consist of a four-stranded (3¨sheet surrounded by three a-helices, with a CXXC
redox active-site
motif. The assembly of various Trx modules has been used to build the
different thiol
oxidoreductases found in prokaryotic and in eukaryotic organisms. In the
bacterial periplasm, the
proteins are kept in the appropriate oxidation state by a combined action of
the couples DsbB-
DsbA and DsbD- DsbC/DsbE/DsbG. Protein expression systems are well known in
the art and
commercially available. Also preferred are E. coli expression strainsthat
expresses constitutively
a chromosomal copy of the disulfide bond isomerase DsbC. DsbC promotes the
correction of mis-
oxidized proteins into their correct foini. Cytoplasmic DsbC is also a
chaperone that can assist in
the folding of proteins that do not require disulfide bonds.
Recombinant bacteria containing expressible protein sequences, wherein an f-
Met that is
present at the N-terminus of the newly expressed protein is enzymatically
removed. Preferred host
cells include, but are not limited to, cells genetically engineered to shift
the redox state of the
cytoplasm to a more oxidative state, that contain and express an inducible MAP
gene. Preferred
cells are prokaryotes such as E. coli expression systems, Bacillus subtillis
expression and other
bacterial cellular expression systems. Preferably the cells contain a protein
expression system for
expressing foreign or non-native sequences. Also preferable, the sequences to
be expressed are
comprised of an expression vector which contains one or more of an inducible
promoter (e.g.,
inducible preferably with specific media), a start codon (e.g., ATG), a
ribosome binding site,
and/or a modified sequence between ribosome binding site and ATG starting
codon, or between
start codon and the sequence to be expressed. Preferred modified sequences or
spacer sequences
include, for example, a number of nucleotides more or less than 9 (e.g.,
between 7 and 12
nucleotides). and preferably not 9 nucleotides.
It has also been surprisingly discovered that recombinant cells can be
developed containing
additional proteases that effectively cleave one or more different pre-
proteins and/or pre-pro-
proteins from the inactive to the active configuration. These proteins are
generally referred to as
zymogens (e.g., proenzymes) requiring post-translational modifications.
Protein precursors are
often used by a cell when the active protein is harmful, but needs to be
expressed. By integrating
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these proteins into a recombinant cell, expression can be achieved safely and
cost-effectively, and
in large quantities. A protease gene which expresses a protease that performs
the specific cleavage
from inactive to active can be integrated into the cellular genome or
transformed with a vector
containing the protease gene of interest, all as described herein. The
introduced protease gene can
be placed under the control of a promotor in common with the recombinant gene
to be expressed
and collected, and the protease gene which clips of the methionine, or the
protease gene may have
a different promotor. Similarly, the gene may be inducible, either separately
or induced basically
simultaneously the recombinant gene to be expressed and collected, and the
protease gene which
clips of the methionine. Preferably, when expression of the recombinant
protein is sufficiently
done, the second protease would be activated and process the pro-protein to an
active state.
Preproteins where this would be effective in both save both time and cost
include, but are not
limited to pro-insulin to insulin, pro-insulin-like proteins to insulin-like
proteins, prorelaxin to
relaxin, proopiomelanocortin to opiomelanocortin, pro-enzymes to enzymes, and
prohormones to
hormones, and also removal of signal peptides, leader sequences, tags, etc.,
from a protein. In
general, the protease introduced will be specific to the protein to be
cleaved. Additional proteins
which could be efficiently produced in this was include, but are not limited
to angiotensinogen,
trypsinogen, chymotrypsinogen, pepsinogen, proteins of the coagulation system
(e.g.,
prothrombin, plasminogen), proteins of the compliment system, procaspases,
pacifastin,
proelastase, prolipase, procarboxypolypeptidases. In addition, certain genes
can be modified to
include a portion (e.g., leader or tag or internal sequence), that allows the
protein to be expressed
in an inactive form, which is only transformed into an active form upon being
cleaved with a
protease whose gene has also be introduced to the cell and subsequently
activated.
By way of example, the gene of interest is inserted into the genome with a
different
promoter. The cell is induced to express that gene, which has disulfide bonds,
and also the
methionine peptidase, which trims off the methionine. Once a suitable amount
of trimmed protein
is produced in the cytoplasm, the second promoter is induced which processes
the protein to its
final or active form. Expression of active protein such as trypsin during
growth would chew up a
lot of needed proteins in the cytoplasm and interfere with expression of the
recombinant protein.
This approach would avoid the need for in vitro processing of the expressed
pro-protein.
Another embodiment of the invention is directed to recombinant protein that is
expressed
in E. coli or another host cell using an expression vector with an inducible
promoter and/or a
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modified sequence between ribosome binding site and ATG starting codon, cells
wherein an f-met
that is present at the N-terminus of the recombinant protein that is
enzymatically removed.
Preferably, the expression vector includes the lactose/IPTG inducible
promoter, preferably a tac
promoter, and the sequence between ribosome binding site and ATG starting
codon.
Another embodiment of the invention comprises an expression construction of
nucleotide
or amino acids sequences and with or without a regulatory region. Regulatory
regions regulate
protein expression by adding one or more sequences that promote nucleic acid
recognition for
increased expression (e.g., start codon, enzyme binding site, translation or
transcription factor
binding site) or for inhibited expression (e.g., operators). Preferably, a
regulatory element of the
invention contains a ribosome binding site with a start codon upstream of and
with a coding
sequence that differs from the coding sequence of the recombinant protein.
Another embodiment of the invention is directed to proteins and peptides as
well as
portions and domains thereof, that can be manufactured according to the
methods disclosed herein.
Proteins and peptides comprise, but are not limited to, for example, those
proteins and peptides
that can be cytoplasmically expressed without leader or tag sequences and at
commercially
significant levels according to the methods disclosed and described herein.
Preferably, these
proteins and peptides show proper folding upon expression in recombinant cells
of the invention.
Recombinant cells of the invention preferably show reduced activity of one or
more disulfide
reductase enzymes, preferable reduced activity of less than five disulfide
reductase enzymes,
preferable reduced activity of less than four disulfide reductase enzymes, and
preferable reduced
activity of less than three disulfide reductase enzymes. Preferably expression
of the proteins and
peptides is increased in recombinant cells of the invention but may be not
reduced or not
significantly reduced compared with expression in recombinant cell that does
not have reduced
activity of one or more disulfide reductase enzymes. Proteins and peptides
that can be expressed
in the methods disclosed herein include, but are not limited to, for example,
tetanus toxin, tetanus
toxin heavy chain proteins, diphtheria toxoid, CRM, tetanus toxoid,
Pseudomonas exoprotein A,
Pseudomonas aeruginosa toxoid, Borcletella pertussis toxoid, Clostridium
perfringens toxoid,
Escherichia coli heat-labile toxin B subunit, Neisseria meningitidis outer
membrane complex,
Hemophilus influenzae protein D, Flagellin Fli C, Horseshoe crab Haemocyanin,
and fragments,
derivatives, and modifications thereof.
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Another embodiment of the invention is directed to portions and domains of
proteins that
are expressed thereof, fused genetically or by chemical modification or
conjugation (e.g.,
carbodiimide, 1-cyanodimethylaminopyridinium tetrafluoroborate (CDAP)) with
another
molecule. Preferred other molecules are molecules such as, hut not limited to,
other proteins,
peptides, lipids, fatty acids, saccharides and/or polysaccharides, including
molecules that extend
half-life (e.g., PEG, antibody fragments such as Fc fragments), stimulate
and/or increase
immunogenicity, or reduce or eliminate immunogenicity.
Many proteins contain an N-terminal serine or threonine or may be genetically
expressed
with an N-terminal serine or threonine. An N-terminal scrinc or threonine can
be selectively
activated making it useful for conjugation. The presence of an N-terminal
Methionine would block
the ability of these amino acids to be selectively activated. The method
described in this patent
allow for the N-terminal Methionine to be cleaved allowing for the protein to
be produced with
the desired N-terminal amino acid. Typical conjugation partner molecules
include, but are not
limited to polymers such as, for example, bacterial polysaccharides,
polysaccharides derived from
yeast, parasite and/or other microorganisms, polyethylene glycol (PEG) and PEG
derivatives and
modifications, dextrans, and derivatives, modified, fragments and derivatives
of dextrans. One
example of a conjugated compound is PEGASYS (peginterferon alfa-2a). Other
polymers, such
as dextran, also increase the half-life of proteins and reduce imniunogenicity
of the conjugate
partner. Polymers may be linked randomly or directed through site specific
conjugation such as,
for example, by modification of N-terminal serine and/or threonine. Also,
modifications may be
used that selectively oxidize chemical groups for site specific conjugation.
Another embodiment of the invention is directed to methods of producing a
peptide
containing a domain, fragment and/or portion comprising: expressing the
peptide from a
recombinant cell containing an expression vector that encodes the peptide,
wherein the
recombinant cell has a reduced activity of one or more disulfide reductase
enzymes and the
expression vector contains a promoter functionally linked to a coding region
of the peptide,
wherein the one or more disulfide reductase enzymes comprises one or more of
an oxidoreductase,
a dihydrofolate reductase, a thioredoxin reductase, or a glutathione
reductase; and isolating the
peptide expressed, wherein the peptide expressed is soluble and wherein the
protein or peptide is
expressed with an f-met at the N-terminus that is removed by a peptidase that
is also expressed
within the recombinant cell. Preferably the expression vector contains a
ribosome binding site, an
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initiation codon, and, optionally, an expression enhancer/repressor region.
Preferably the
recombinant cell has a reduced activity of only one disulfide reductase
enzyme, only two disulfide
reductase enzymes, or two or more disulfide reductase enzymes. Preferably the
reduced activity
of the disulfide reductase enzymes results in a shift the redox status of the
cytoplasm to a more
oxidative state as compared to a recombinant cell that does not have reduced
activity of one or
more disulfide reductase enzymes. Preferably the recombinant cell is an E.
coil cell or a derivative
or strain of E. coll. Preferably the soluble peptide expressed comprises a
natively folded protein
or domain of the protein. The promoter may be a constitutive or inducible
promoter, whereby
expression comprises inducing the inducible promoter with an inducing agent.
Preferred inducing
agents include, for example, lactose (PLac), isopropyl 3-D-1-
thiogalactopyranoside (IPTG),
substrates and derivative of substrates. In one preferred embodiment, the
genome of the
recombinant cell contains an additional gene that preferably contains a coding
region for a
peptidase that preferably acts upon and selectively cleaves the peptide or
protein expressed from
the expression vector. Preferably the recombinant protein expression vector
contains the same or
a different inducible promoter as the MAP gene that has been inserted into the
gcnome. The
additional gene and the gene in expression vectors may be induced together
with the same inducing
agent, or with different inducing agents, optionally at different times
depending on the promoters.
Preferably the peptidase acts on and cleaves the peptide co-expressed with the
peptidase.
Preferably the peptide expressed is conjugated with a polymer such as, for
example, dextran, a
bacterial capsular polysaccharide, polyethylene glycol (PEG), or a fragment,
derivative or
modification thereof. Preferably the peptide expressed is coupled with a
polymer which includes,
for example, a polysaccharide, a peptide, an antibody or portion of an
antibody, a lipid, a fatty
acid, or a combination thereof.
Another embodiment of the invention comprises conjugates of proteins expressed
and
cleaved according to the disclosures herein including fragments, domains, and
portions thereof as
disclosed and described herein.
Another embodiment of the invention comprises fusion molecules of proteins
included
fragments, domains, and portions thereof as disclosed and described herein.
Another embodiment of the invention comprises a vaccine of proteins included
fragments,
domains, and portions thereof, as disclosed and described herein.
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The following examples illustrate embodiments of the invention but should not
be viewed
as limiting the scope of the invention.
Example 1. Insertion of MAP gene into various loci of the bacterial genome.
Efficiently removal of the N-terminal Methionine in overexpressed
intracellular
recombinant proteins was achieved by inserting a MAP gene with a promoter,
preferably inducible,
into the E. coli genome. In this way a permanent cell line was created that
expresses the MAP
gene under the control of an inducer. A recombinant gene is cloned into the
cell, also under control
of an inducer. Thus, the MAP gene can be expressed at the desired time to
efficiently cleave the
N-terminal Methionine from the expressed recombinant protein. Many MAP enzymes
other than
E. coli MAP are known or have been devised. MAP from other species may have
different
selectivity for the adjacent amino acid. Some MAPs have been genetically
altered to be less
stringent in their requirement for a non-bulky amino acid adjacent to the N-
terminal Methionine.
The insertion of one or more of these MAPs with an inducible promoter into the
E. coli genome
would expand the range of N-terminal sequences which could be efficiently
processed.
Inserting a recombinant gene into a genome may disrupt the L. coli genome
structure,
possibly impairing cell growth. A safe way to insert the MAP gene into the If,
coli genome is to
use a viable strain from which a gene has been deleted and to substitute in
the MAP gene for the
deleted gene. In this way, by replacing a gene for a gene, the probability of
disruption of the
genome can be reduced. Two strains of E. coli are widely used to manipulate
genes and express
recombinant proteins: the K12 strain and the B strain. E. coli strains,
including the K12 and B
strains, can be used as the host cell for the insertion of the MAP gene.
Insertion at the wrong site might be lethal to the bacteria, such as an
insertion deletion in
an open reading frame of an essential gene or at a site which disrupts control
elements. Three
illustrative protocols to insert the MAP gene, with an inducible promoter and
a terminator are:
(1) Insertion of the MAP gene upstream or downstream of an already defined
gene. For
example, the recombinant MAP gene can be inserted downstream of the endogenous
MAP gene.
The E. coli MAP gene has been studied and the gene structure has been defined.
Insertion of the
recombinant gene downstream will not disturb the expression of other gene.
(2) Insertion of the MAP gene into a gene locus that has been previously
deleted. The
creation of the Gor/Met cell line is an example of the insertion of the MAP
gene into the site of a
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deleted gene. Gor- cells were created by deleting the Gor gene in BL21 cells.
The Gor/Met cell
line was created by insertion of the MAP gene into the Gor locus of BL21 Gor-
cells.
(3) Insertion/replacement of a nonessential gene. As an example of the third
method, the
MAP gene is inserted to replace the T7 RNA polymerase in BL21(DE3) cells. BL21
(DE3)
encodes a very active T7 RNA polymerase in the DE3 fragment which can
transcribe recombinant
genes under the control of T7 promoter. T7 polymerase gene can be replaced by
MAP gene if the
recombinant gene is transcribed by intrinsic RNA Polymerase under the control
of T5 promoters.
Site directed mutagenesis techniques are required for the above-mentioned
insertion site
selection. Many methods are known to manipulate the bacterial genome and two
examples are
disclosed here. The most common method for the insertion/deletion of a gene is
Red
Recombineering. This technique has been used widely for mutations in bacteria
genomes as well
as eukaryotic genomes and starts with using PCR to introduce short sequences
of DNA
complementary to upstream and downstream of the selected site of insertion
flanking the gene of
interest. The PCR product is then electroporated into E. coli that has already
expressed red
recombinase in a previously transformed temperature sensitive vector. The red
recombinase aids
in the homologous recombination which inserts the gene at the selected site.
Red recombinase can
be removed by growing the bacteria at 42 C since Red recombinase gene is on a
temperature
sensitive plasmid.
To enhance the selection for positive clones, a marker gene can be introduced
and later
removed after positive clones are confirmed. In one method, a flippase
recognition signal is
introduced to flank a marker gene, such as an antibiotic gene, that cloned
downstream of inserted
gene. The PCR product that was used for gene insertion will then include the
marker gene. After
the recombination event occurred, the marker gene can be used for positive
clone selection. Once
the positive clone is confirmed, flippase expression is introduced to the
bacteria to remove the
marker gene between two flippase recognition sites. This kind of insertion is
marked with a scar
that contains flippase recognition sequences at the insertion site. 'Ellis
method was used to create
the Gor/Met E. coli strain by inserting the Met gene into the deleted Gor gene
and is described in
Example 2 below.
CRISPR technology can be used in E. coli when combined with Red
recombineering. In
the CRISPR-assisted red recombineering, two plasmids and one oligos are
utilized. One plasmid
encodes constitutively expressed Red recombinase and Cas9 protease. Another
encodes the
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CRISPR guide RNA with the insertion site cloned in. These two plasmids have
two compatible
replication-of-origins. The oligo is made to contain the gene of interest
flanked by the insertion
site sequences. E. coli cells are transformed with these three elements and
the surviving colonies
should he the one that have the gene inserted: Cas9 protease will hind with
CRISPR guide RNA
to scan for the insertion site. Once the insertion site is located, Cas9 will
cleave its double stranded
DNA and allow the red recombinase to come in to perform homologous
recombination between
the cleaved site and the oligo with protein of interest. Those colonies with
original sequence will
be recognized and eliminated. Only the ones have the gene of interest will
survive. The CRISPR
assisted Red recombineering has a 65% success rate, while the other 35% comes
from the failure
of Cas9 to locate the insertion site. Thus, screening for positive clones is
fast and straightforward.
Example 2. Construction of an E coil cell strain with a gene replacement for
the
cytoplasmic expression of recombinant proteins without N-terminal Methionine
The construction of E. coil cell strain BL21 Gor- is described in U.S. Patent
Nos.
10,093,704 and 10,597,664. The strains described have the Gor gene deleted and
an oxidative
cytoplasm, such that proteins arc expressed cytoplasmically, with properly
folded disulfide bonds,
and at high levels. To these stains, an extra E. coli MAP gene was inserted
into the Gor gene locus
and the new strain called Gor/Met E. coll. A Tac promoter (with a Lac
operator) was added
upstream of the MAP gene, so that expression of the MAP gene can be regulated
by the timing of
IPTG addition. This was accomplished as follows: An additional E. coli MAP
gene was inserted
into genome by homologous recombination. In the case of Gor/Met cells, the red
recombinase
system was used to aid in the insertion of the MAP gene into the Gor locus in
BL21 Gor- cells.
The MAP gene was PCR amplified from the BL21 Gor- genome and put under the
control of a
Tac promoter. The gene was then cloned downstream of a chloramphenicol
acetyltransferase
(CAT) gene flanked by two short flippase recognition target (FRT) sequences.
The MAP and CAT
gene together formed a transfer gene cassette. PCR was used to introduce fifty
bases each of
sequences flanking upstream and downstream of the original Gor locus into the
transfer cassette
upstream and downstream, respectively. The final PCR product was used to
transform to BL21
Gor- cells previously transformed with Red recombinase. The expression of Red
recombinase in
the cell accelerated the homologous recombination of the sequences flanking
Gor locus with that
of transfer cassette. Bacterial colonies that were resistant to
chloranaphenicol were confirmed to
have transfer cassette insertion. Confirmed bacteria were subsequently
transformed with Ilippase
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gene whose expression recognized FRT sequence flanking the CAT gene and
clipped the gene
away to leave the MAP still inserted. Thus, the Gor/Met cell line has MAP gene
inserted at the
Gor locus, without disturbing other parts of the genome. This cell line was
deposited strain and
deposited with the American Type Culture Collection as Deposit No. PTA-126975
on February
09, 2021. The results of sequencing using primers designed to flank the Gor
locus confirmed the
successful insertion of the MAP gene.
To demonstrate the functionality of the strain, several genes were expressed
in BL21
Gor/Met cells and confirmed to efficiently cleave the N-terminal Methionine to
produce the native
protein.
Example 3. Expression of CRM197 in Gor/Met E. coli
CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that
contains a
single amino acid substitution G52E. Like DT, CRM197 has two disulfide bonds.
One disulfide
joins Cys186 to Cys201, linking fragment A to fragment B. A second disulfide
bridge joins
Cys461 to Cys471 within fragment B. CRM197 is commonly used as the carrier
protein for
carbohydrate-, peptide- and haptcn-protein conjugates. As a carrier protein,
CRM197 has a number
of advantages over diphtheria toxoid as well as other toxoided proteins.
Although CRM197 has been produced in the original host Corynebacterium, a slow
growing
bacteria with a doubling time of hours instead of minutes, yields are low,
typically <50mg/L.
Corynebacterium strains have been engineered to produce CRM197 at higher
levels (e.g., see U.S.
Patent No. 5,614,382). CRM197 has also been expressed in a strain of
Pseuclornonas fluorescens
at a high level. However, production of CRM197 in a strain that is at a BL1
safety level and is
inexpensive to culture and propagate would be advantageous. Expression of
soluble, properly
folded intracellular CRM197 in BL21 Gor- strain has been successful, with >2g
CRM197 per liter
fermenter cell culture. However, the majority of the CRM197 produced was found
to have N-
terminal f-methionine. The CRM197 gene with a lac promoter was cloned into the
Gor/Met E coli
strain (e.g., see U.S. Patent Nos. 10,093,704 and 10,597,664 for the gor-
strain). Thus, both the
MAP gene and the recombinant CRM197 gene were under the control of the same
tac promoters,
capable of being expressed simultaneously upon IPTG induction.
Expression of CRM197 in BL21 gor- and BL21 Gor/Met E. coli were compared.
Similar
yields, ¨ 2 g/L were found for both strains, indicating that co-expression of
the MAP gene did not
significantly affect the expression of the CRM197. Purified CRM197 from BL21
gor- and BL21
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Gor/Met strains were analyzed by MALDI-TOF mass spectrometry and the results
summarized in
Table 1. Lot NO21p114 was expressed in the Gor- strain and Lot NO21p221 was
expressed in
the Gor/Met strain. CRM197 expressed in 13L21 Gor- contained N terminal Met
whereas CRM197
expressed in Gor/Met E. coli did not, demonstrating that the method described
in this invention
was successful.
Table 1
Non-reduced CRM197 (n=3, 1SD)
Species Theoretical Observed
NO21p114 (gor-) NO21p221(GorMet)
CRM (w/out Met) -58,409 Da 58,411.6 0.4 Da 58,411.4
0.0 Da
CRM (with Met) -58,540 Da 58,542.2 0.1 Da not
observed
Example 4. Expression or cytokine IL10 from Epstein-Barr virus in the Gor/Met
strain.
The IL10 gene, derived from the Epstein-Barr virus, was cloned and expressed
as a soluble
intracellular protein in Gor/Met E. coli. A metal affinity tag was included on
the C-terminal to
facilitate purification. The IL10, purified by IMAC and ion exchange
chromatography was
subjected to mass spectrometry analysis to determine the sequence of the N-
terminal peptide.
Following enzymatic digestion with trypsin, the sample was analyzed by LC-
MS/MS, which found
that the protein did not have an N-terminal methionine.
The procedure was carried out using the following protocol: The sample was
digested with
trypsin and analyzed by LC-MS/MS on a LTQ Orbitrap Velos (ThermoFisher
Scientific, Bremen,
Germany), interfaced with a Proxeon 1200 nanoLC (Proxeon Biosystems). The
chromatography
was performed on a 75 tm i.d. Self-Pack PicoFrit fused silica capillary column
15 cm in length
(New Objective, Woburn, MA). The stationary phase was a reverse-phase C18
Jupiter column (5
R' , 300 A) (Phenomenex, Torrance, CA). Mass resolution was set to 30 000 for
parent mass
determination in MS mode and to 7 500 for acquisition of the fragmentation
spectra in MS/MS
mode. MS/MS spectra obtained during the LC-MS/MS run were submitted to a
Mascot search
against the expected protein sequence. Carbamidomethyl (C) was selected as
Fixed modifications,
and Oxidation (M) was selected as Variable modifications. The objective was to
retrieve from
the digest solution the peptide corresponding to the first tryptic cleavage.
This peptide, on the
submitted sequence, would include the Arginine on position 13. If Methionine
was present on the
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N-terminus, the result would be amino acid sequences 1 to 13 (MTDQCDNFPQMLR;
SEQ ID
NO: 1), while if Methionine would be absent, the results would be amino acid
sequences 2 to 13
(TDQCDNFPQMLR; SEQ ID NO: 2).
Table 2
1 MTDQCDNFPQ MLRDLRDAFS RVKTFFQTKD ELDNLLLKES LLEDFKGYLC
51 CQALSEMIQF YLEEVMPQAE NQDPEIKDHV NSLGENLKIL RLRLRRCHRF
101 LPCENKSKAV EQIKNAFNKL QEKGIYKAMS EFDIFINYIE AYMTIKARGH
151 HHHHH ( SEQ ID NO: 3)
The amino acids underlined and bolded in Table 2 identify the sequences
identified by
Mascot from the MS/MS sequencing data. This signifies that ions were found to
confitm the 2-13
sequence (no Methionine on the N-terminus) and that no ions were found for the
1-13 sequence
(Methionine on the N- terminus).
The 2-13 sequence (without a Methionine on the N-terminus) was confirmed by
the
presence of the following ions in the MS trace: 762.8 m/z (+2), 508.9 m/z
(+3), 770.8 in/z, (+2), and
their corresponding fragmentation pattern from the MS/MS trace. The three
identified ions all
contain an alkylated cysteine but the ion at 770.8 nilz also contains an
oxidized methionine
(position 11 on the submitted sequence). In conclusion, the IL10 expressed in
Gor/Met was
produced without the N-terminal Methionine.
Example 4. Expression of a genetically detoxified tetanus toxin (8MTT) in
Gor/Nlet E. coli
Tetanus toxin is known as one of the most potent toxins for humans and
referred to as a
spasmogenic toxin, or TeNT. The LD50 of this toxin is measured to be
approximately 2.5-3 ng/kg.
Tetanus toxin is produced by Clostridium tetani, an anaerobic bacillus
normally found in soil, as
a single polypeptide chain that is post translationally cleaved by a trypsin-
like protease into two
chains to form the active protein. The light chain (LC), a 50kDa domain,
contains a N-terminal
endopeptidase, the heavy chain (HC) contains a 50kDa receptor binding domain
on C terminus
(HCC) and a 50 kDa LC translocation domain is located on the N terminus (HCN).
The two chains
are connected by a single disulfide bond. Tetanus toxin enters peripheral
motor neurons by binding
to gangliosides and synaptic proteins on their surface through the C-terminal
domain of the heavy
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chain (HCC). The toxin traffics to the soma and to synapses of interneurons in
the central nervous
system, and transcytoses and enters inhibitory neurons in synaptic vesicles.
In the inhibitory
neurons, the heavy chain's translocation domain (HCN) undergoes a pH-mediated
confol __ national
change and transports the LC through the membrane of synaptic vesicles into
the cytoplasm where
LC is released into the cell cytosol and cleaves vesicle-associated membrane
protein 2 (VAMP2),
a vesicle soluble NSF attachment protein receptor (SNARE). VAMP2 cleavage in
inhibitory
neurons blocks neurotransmitter exocytosis, preventing release of inhibitors
of neuromuscular
synapse function, leading to continued neuromuscular activation and spastic
paralysis.
Chemically inactivated tetanus toxoid (TTxd), formed by treating the toxin
with
formaldehyde, is used as an effective vaccine against tetanus. TTxd is also
used as a conjugate
vaccine carrier for polysaccharide antigens. Conjugated vaccines using TTxd as
the carrier protein
include vaccines against Haemophilus influenzae type b and Neisseria
meningiditis. However,
TTxd has many of its amines, used for conjugation, blocked by the toxoiding
process.
Furthermore, TTxd is a heterogeneous product and contains aggregates, along
with Clostridium
and media contaminants. TTxd vaccine needs to be further purified for use in
conjugate vaccines.
More important, the production and purification of TT from the Clostridium is
time consuming
and costly. A genetically inactivated homogeneous recombinant Tetanus toxin,
produced in a low-
cost host like E. coli, would be desirable.
Different strategies of producing inactivated recombinant TT proteins as
vaccines or carrier
proteins have been explored. One is the use of heavy chain fragments (TTHC).
Another is the use
of genetically inactivated tetanus toxin (U.S. Patent Application Publication
No. 2020/03841201).
TTHC is part of the TT that does not carry the catalytic domain. Neutralizing
antibodies against
the TTHC subunit vaccine was claimed to outperform full toxoid vaccine
antibodies. TTHC was
expressed at high levels (>400 mg/L) in the BL21 Gor- system (e.g., see U.S.
Patent Nos.
10,597,664 and 10,093,704).
8Mr1"f is a genetically detoxified tetanus toxin ('IT) with 8 amino acid
mutations. Like
tetanus toxin, 8MTT has 5 disulfide bonds. The LD50 is more than 50 million-
fold less toxic than
native TT. 8MTT vaccination elicited a strong immune response IgG antibody
response in mice,
is a lead candidate for a new tetanus vaccine and, has great potential to be
used as a conjugate
vaccine carrier protein, similar to the widely used CRM197. 8MTT was
originally cloned into the
pET28 expression vector and expressed in BL21(DE3) cell with a His tag
attached to facilitate the
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purification. The expression was about 10 mg/liter in the shaker flasks. To
produce protein
without the tag and without the N-terminal Methionine, 8MTT gene was subcloned
into an
expression vector with the tac promoter (with lac operator) and T7 tel
and then expressed
in BL21 Gor/Met cells in a fed-batch fermenter. The expressed M8TT protein was
found to he
soluble and could be purified at more than 500mg per liter. Using a
combination of anion exchange
column, HIC column and TFF diafiltration/concentration M8TT was purified to
more than 99%
purity. The purified M8TT was analyzed by MALDI-ISD (Matrix Assisted Laser
Desorption/Ionization - In Source Decay) to obtain terminal fragmentation. ISD
allowed for the
identification of a ladder of N-terminal fragments and confirmed that the
sequence did not have an
N-terminal Methionine and the first residue of the sequence was the expected
Proline. Thus, the
Gor/Met E. coli strain efficiently expressed large quantities of soluble M8TT
without an N-
terminal methionine.
Example 5. CRM197 mutant with N-terminal serine
The CRM197 gene containing an N-terminal serine (CRM-Ser) was cloned into the
Gor/Met
E. coli strain and grown and expressed in a biorcactor. Without any
optimization of fermentation
conditions >1 g/L of soluble CRM-Ser was expressed, showing that expression of
the protein was
excellent. The cells were harvested and CRM-Ser purified. The CRM-Ser was
analyzed by
MALDI-ISD as described in Example 4. ISD allowed for the identification of a
ladder of N-
terminal fragments and confirmed that the sequence did not have an N-terminal
Methionine and
the first residue of the sequence is the expected Serine. Thus, the Gor/Met E.
coli strain efficiently
expressed large quantities of soluble CRM-Ser without an N-terminal
methionine. The Seiine can
be selectively oxidized and used for conjugation.
Example 6: Possible MAP genes to insert in bacterial genome.
Removal of N-terminal Methionine by MAP can be important for proper function
and
stability of proteins. Most of the recombinant proteins expressed in E. coli
still have methionine
starting codon on N terminus even though the intrinsic MAP is active.
Insufficient MAP or its
cofactors may be present when overexpressed recombinant proteins are produced.
To ensure the
processing of the N terminal methionine in recombinant proteins, extra MAP
genes are inserted
under strong, inducible promoters to facility methionine cleaving process when
necessary.
1. E. coli MAP gene
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Gor/Met cell is an example to insert an extra E. coli MAP gene in bacteria
genome and
under the same inducible promoter as that of the recombinant gene on the
expression vector. When
not induced, this MAP gene remains silent and the bacteria propagate with no
burden of extra gene
expression. As only the recombinant protein is induced to be expressed that
extra MAP protein
are also induced, MAP protein can be designed to be turned on as needed. A
potential drawback
of this system is that not all proteins with N terminal methionine can be
efficiently cleaved by E.
coli MAP. E. coil MAP works when a small amino acid (G, A, S. C, P. T, V) (P1'
position) is
adjacent to N-terminal Methionine. Preferably the P2' position (the amino acid
C terminal to P1)
of amino acid is not proline. To process those proteins with bulky amino acids
next to the N-
terminal Methionine, other MAP genes with different P1 and P2 amino acid
requirements maybe
inserted instead.
2. Two different MAP gene in tandem in different inducible promoters
Yeast genes are processed by two MAP genes. Yeast MAP1 and MAP2 exhibit
different
cleavage efficiencies against the same substrates in vivo. Both MAPs were less
efficient when the
second residue was V, and MAP2 was less efficient than MAP1 when the second
residue was G,
C, or T. Humans also have two MAPs: MAP1 and MAP2. They can both process
proteins
containing A, C, G, P, or S at the P1' position. When the P1' residue is T or
V. the N-terminal Met
removal is primarily catalyzed by MAP2 and the extent of cleavage depends on
the sequence at
P2'¨P5' positions. When the P2' residue is not A, G, or P. the N-terminal
processing is expected
to be complete. When A, G, or P is the P2' residue, Methionine removal is
either incomplete or
does not occur. Since different MAP has different substrate specificity, two
or more MAP genes
from different or same species may be able to cover Methionine removing
processing from more
recombinant proteins. The (for example inducible) promoters that control the
gene transcription
maybe different so that two MAP genes can be turned on at different time, or
one on/one off,
depending on the need.
3. Insert a mutated MAT gene that is capable to cleave all IN terminal
methionine without
restriction on the amino acid followed.
The current MAPs disfavor some protein's N terminal structures and will not
catalyze the
removal of their N terminal Methionine. A universal rule that predicts whether
the initiating
Methionine will be process by MAPs is based on the size of amino acid at the
P1' position. In
general, if amino residues have a radius of gyration of 1.29 A or less,
Methionine is cleaved. For
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human MAPs, they have even more stringent requirement for substrates have
acidic residues at
the P2' an P5' position. To expand the substrate specificity of the existing
MAPs, an E. coli MAP
gene was mutated so that its product can cleave 85-90% of N terminal
methionine on proteins
listed in the protein database. This MAP has three mutations in its substrate
binding pocket, thus
allows removal of N-terminal Methionine from proteins with not only small
amino acid but also
bulky or acidic amino acid (e.g., M, H, D, N, E, Q, L, I, Y and W, at P2'
position). These enzymes
can also cleave the amino acid at the P1' position if amino acid residue at
P2' position is small.
Insertion of this MAP gene processes proteins with broader N terminal
structure. Again, adding
an inducible promoter will be beneficial to control the activity of this
powerful mutant gene
expression.
Example 7: Proteins capable of produced in the Gor/Met cell line without
disulfide bond
Although the Gor/Met cell strain was developed to express disulfide bond
proteins without
an N-terminal Methionine, proteins without disulfide bond can be expressed in
Gor/Met cells
without an N terminal methionine. These proteins can also be expressed in E.
coli with a Met but
not necessarily be gor-. An example of such a protein is Staphylococcus
protein A (SPA) which
is widely used in antibody purifications. SPA is a 42 kDa protein originally
found in the cell wall
of the bacteria Staphylococcus aureus. This protein is composed of five
homologous Ig-binding
domains that can bind proteins from many mammalian species, particularly IgGs
and binds the
heavy chain within the Fe regions of most immunoglobulins and within the Fab
regions of the
human VII3 family. SPA does not contain any disulfide bonds. Commercially, SPA
comes from
two sources: Staphylococcus aureus mutant strains that contains lesions in the
cell wall where
SPAs are secreted (Sigma), E. coli that expresses SPA as a recombinant
protein, either intra- or
extra-cellularly (Sigma-Aldrich, SinoBiological, ThermoFisher and DeNovo
Biopharma, and
others). SPA production can be at high levels in Pichia pastoris. The majority
of SPA are
expressed intracellularly E. coll. Since the mature SPA sequence starts with
Alanine and not
Methionine, for SPA to be produced intracellularly, either a methionine needs
to be added to the
gene at the N-terminus or it needs to be expressed at the C-terminus of a
fusion protein which can
be cleaved to release the mature protein in vitro. The former is not the true
form of mature protein,
the latter is not cost and time efficient.
The expression of SPA is efficient in bacterial strains disclosed herein that
have an
inducible MAP gene insertion. SPA is expressed in E. coli strains in high
quantity, and functions
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similarly as in the Gor/Met cell line and can also be expressed in a MAP cell
line. The advantage
of using MAP cell line over other cell line is that the f-methionine can be
cleaved at will. The
inducible promoter of MAP gene can be expressed at the same time when SPA is
expressed or
anytime afterwards, by design. No in vitro manipulation is required to remove
the N-terminal
Methionine.
Other embodiments and uses of the invention will he apparent to those skilled
in the art
from consideration of the specification and practice of the invention
disclosed herein. All
references cited herein, including all publications, U.S. and foreign patents
and patent applications,
are specifically and entirely incorporated by reference. The term comprising,
where ever used, is
intended to include the terms consisting and consisting essentially of.
Furthermore, the terms
comprising, including, and containing are not intended to be limiting. It is
intended that the
specification and examples be considered exemplary only with the true scope
and spirit of the
invention indicated by the following claims.
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